Inactivation of Heart Dihydrolipoamide Dehydrogenase by Copper Fenton Systems. Effect of Thiol Compounds and Metal Chelators

Copper Fenton systems (Cu(II)/H₂O₂ and Cu(II)/Asc) inactivated the lipoamide reductase and enhanced the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). Cupric ions alone were less effective. As a result of Cu(II)/H₂O₂ treatment, the number of titrated thiols in LADH decreased from 6 to 1 per subunit. NADH and ADP (not NAD⁺ or ATP) enhanced LADH inactivation by Cu(II). NADH also enhanced the effect of Cu(II)/H₂O₂. Dihydrolipoamide, dihydrolipoic acid, Captopril, acetylcysteine, EDTA, DETAPAC, histidine, bathocuproine, GSSG and trypanothione prevented LADH inactivation. 100 μM GSH, DL-dithiothreitol, N-(2-mercaptopropionylglicine) and penicillamine protected LADH against Cu(II)/Asc and Cu(II), whereas 1.0 mm GSH and DL-dithiothreitol also protected LADH against Cu(II)/H₂O₂. Allopurinol provided partial protection against Cu(II)/H₂O₂. EthanoI, mannitol, Na benzoate and superoxide dismutase failed to prevent LADH inactivation by Cu(II)/H₂O₂ or Cu(II). Catalase (native or denaturated) and bovine serum albumin protected LADH but that protection should be due to Cu binding. LADH inhibited deoxyribose oxidation and benzoate hydroxylation by Cu(II)/H₂O₂. It is concluded that site-specifically generated HO, radicals were responsible for LADH inactivation by Cu(II) Fenton systems. The latter effect is discussed in the context of ischemia-reoxygenation myocardial injury.     

The Hydroxyl Radical Generated by an Iron(II)/EDTA/Ascorbate System Preferentially Attacks Tryptophan Residues of the Protein

Iron(II)/EDTA/ascorbate-mediated oxidative damage to specific amino acid residues (tryptophan) of serum albumin was studied. The active species generated by Fe(II)/EDTA/ascorbate preferred to react with tryptophan residues rather than histidine or other amino acids. The observation of preferential damage to tryptophan residues of the protein was fully suported by a model experiment using a tryptophan analogue. The reaction of Fe(II)/EDTA/ascorbate to the protein was significantly suppressed by mannitol and dimethysulfoxide, suggesting the participation of the hydroxyl radical generated via Fenton’s reaction. The result was supported by the hydroxyl radical assay using 2-deoxyribose.     

Redox cycling of iron by Abeta42

The amyloid cascade hypothesis and oxidative damage have been inextricably linked in the neurodegeneration that is characteristic of Alzheimer's disease. We have investigated this link and sought to suggest a mechanism whereby the precipitation of Abeta42 might contribute to the redox cycling of iron and hence the generation of reactive oxygen species via Fenton-like chemistry. We have shown that the critical step in the auto-oxidation of Fe(II) under the near-physiological conditions of our study involved the generation of H2O2 via O2.- and that Abeta42 influenced Fenton chemistry through aggregation state-specific binding of both Fe(II) and Fe(III). The net result of these interactions was the delayed precipitation of kinetically redox-inactive Fe(OH)3(s) such that Fe(II)/Fe(III) were cycled in redox-active forms over a substantially longer time period than if peptide had been absent from preparations. The addition of physiologically significant concentrations of either Cu(II) or Zn(II) reduced the role played by Abeta42 in the Fe(II)/Fe(III) redox cycle whereas a pathophysiologically significant concentration of Al(III) potentiated the redox cycle in favour of Fe(II) whether or not Cu(II) or Zn(II) was additionally present. The results support the notion that oxidative damage in the immediate vicinity of, for example, senile plaques, may be the result of Fenton chemistry catalysed by the codeposition of Abeta42 with metals such as Fe(II)/Fe(III) and Al(III).     

Inactivation of mitochondrial adenosine triphosphatase from Trypanosoma cruzi by oxygen radicals

Incubation of Trypanosoma cruzi mitochondrial ATPase (Fo-F1) with the xanthine oxidase system (XO), Fenton's reagent (Fe2+ + H2O2) and the ascorbate-Cu system, caused gradual loss of enzyme activity, which increased as a function of incubation time and rate of oxygen radical generation. The essential role of OH. radicals for ATPase inactivation was supported by a) the enzyme protection afforded by superoxide dismutase, catalase and mannitol, when using the XO system; b) the similar effect of mannitol and benzoate with Fenton's reagent; c) the similar effect of catalase, EDTA and histidine with the ascorbate-Cu system; d) the increased rate of ATPase inactivation by 1) the XO system supplemented with chelated iron, and 2) the ascorbate-Cu system supplemented with H2O2. Comparison of oxygen radical generators for their action on membrane-bound (Fo-F1) and soluble F1 revealed that ascorbate-Cu was the most effective one, possibly because of its capability of producing OH. radicals that react preferentially with the enzyme at their formation site.     

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.     

Iron(II)-mimosine catalyzed cleavage of DNA

Mimosine, DNA breaKs, Free Radicals, Fenton Reaction Supercoiled plasmid DNA was treated in vitro with H2O2, DTT and either Fe (II), Fe (II)-EDTA or Fe (II)-mimosine. The rate of DNA break formation was followed by the conversion of the supercoiled form into relaxed-circular and linear forms. In the concentration interval of 0-4 microM Fe (II), Fe (II)-EDTA slowed-down the formation of DNA breaks, while Fe (II)-mimosine enhanced the rate of break formation up to several times. A conclusion is drawn that this enhancement is due to the increased affinity of the Fe (II)-mimosine complex to DNA.     

Thiols can either enhance or suppress DNA damage induction by catecholestrogens

The estrogen metabolites catecholestrogens (or hydroxyestrogens) are involved in carcinogenesis and the development of resistance to methotrexate. This induction of drug resistance correlates with the relative efficiency of catecholestrogens in the generation of reactive oxygen species (ROS) and the induction of DNA strand breaks. Although antioxidants can neutralize ROS, the generation of these reactive species by catecholestrogens can be enhanced by electron donors like NADH. Therefore, this study was undertaken to determine the ability of different thiol agents (GSH, NAC, DTT, DHLA) to either inhibit or enhance the level of DNA damage induced by the H(2)O(2) generating system 4-hydroxyestradiol/Cu(II). Our results show that GSH, DTT, and DHLA inhibited the induction of the 4-hydroxyestradiol/Cu(II)-mediated DNA damage, with GSH showing the best potential. In contrast, the GSH precursor NAC at low concentrations was able to enhance the level of oxidative damage, as observed with NADH. NAC can reduce Cu(II) to Cu(I) producing the radical NAC&z.rad;, which can generate the superoxide anion. However, the importance of this pathway appears to be relatively minor since the addition of NAC to the 4-hydroxyestradiol/Cu(II) system generates about 15 times more DNA strand breaks than NAC and Cu(II) alone. We suggest that NAC can perpetuate the redox cycle between the quinone and the semiquinone forms of the catecholestrogens, thereby enhancing the production of ROS. In conclusion, this study demonstrates the crucial importance of the choice of antioxidant as potential therapy against the negative biological effects of estrogens.     

Studies of the limited degradation of mucus glycoproteins. The mechanism of the peroxide reaction.

The reaction between ovarian-cyst glycoproteins and H2O2 was investigated in the presence of a number of inhibitors and catalysts. Azide and 2H2O were separately found to have little effect, implying that singlet oxygen was not involved. Superoxide dismutase was destroyed by H2O2, but mannitol had no effect: thus generalized attack by OH., whether originating from HO2.- or more directly, is not indicated. The glycoproteins contained trace quantities of Cu and Fe, amounting to about 2 atoms of metal per glycoprotein molecule. Treatment of the glycoproteins with the strong chelator DETAPAC (diethylenetriaminepenta-acetic acid) or Chelex resin eliminated the reaction with H2O2; activity could be restored by addition of Cu2+ or Fe2+ in millimolar quantities. It was concluded that metal-ion catalysis is an essential step in the attack of H2O2 on glycoproteins. Spectroscopic and other evidence showed that Cu2+ (and probably Fe2+) complexes strongly with poly-L-histidine, and implies that the Cu2+ or Fe2+ in the glycoproteins is complexed with some of the histidine residues in the glycosylated backbone. Neither polyhistidine nor polyproline reacted with H2O2 in the absence of metal ions, but small quantities of Cu2+ or Fe3+ caused degradation. This was rapid with polyhistidine, which was converted largely into aspartic acid, but slower with polyproline, where limited conversion into glutamic acid occurs. These findings confirm the original hypothesis that peroxide attack on glycoproteins occurs largely at the histidine residues, with simultaneous peptidolysis. The mechanism most probably involves the liberation of OH. by an oxidation-reduction cycle involving, e.g. Cu+/Cu2+: specificity of attack at histidine is due to the location of the metal at these residues only.     

The ferroxidase activity of yeast frataxin

Frataxin is required for maintenance of normal mitochondrial iron levels and respiration. The mature form of yeast frataxin (mYfh1p) assembles stepwise into a multimer of 840 kDa (alpha(48)) that accumulates iron in a water-soluble form. Here, two distinct iron oxidation reactions are shown to take place during the initial assembly step (alpha --> alpha(3)). A ferroxidase reaction with a stoichiometry of 2 Fe(II)/O(2) is detected at Fe(II)/mYfh1p ratios of < or = 0.5. Ferroxidation is progressively overcome by autoxidation at Fe(II)/mYfh1p ratios of >0.5. Gel filtration analysis indicates that an oligomer of mYfh1p, alpha(3), is responsible for both reactions. The observed 2 Fe(II)/O(2) stoichiometry implies production of H(2)O(2) during the ferroxidase reaction. However, only a fraction of the expected total H(2)O(2) is detected in solution. Oxidative degradation of mYfh1p during the ferroxidase reaction suggests that most H(2)O(2) reacts with the protein. Accordingly, the addition of mYfh1p to a mixture of Fe(II) and H(2)O(2) results in significant attenuation of Fenton chemistry. Multimer assembly is fully inhibited under anaerobic conditions, indicating that mYfh1p is activated by Fe(II) in the presence of O(2). This combination induces oligomerization and mYfh1p-catalyzed Fe(II) oxidation, starting a process that ultimately leads to the sequestration of as many as 50 Fe(II)/subunit inside the multimer.     

Catecholamines Enhance Dihydrolipoamide Dehydrogenase Inactivation by the Copper Fenton System. Enzyme Protection by Copper Chelators

Catecholamines (CAs: epinephrine, norepinephrine, dopamine, L-DOPA, 6-hydroxydopamine) and o-diphenols (DOPAC and catechol) enhanced dihydrolipoamide dehydrogenase (LADH) inactivation by Cu(II) /H₂O₂ (Cu-Fenton system). The inhibition of LADH activity correlated with Cu(II), H₂O₂ and CA concentrations. Similar inhibitions were obtained wit! the assayed CAs and o-diphenols. CAs enhanced HO radical production by Cu(II) /H₂O₂, as demonstrated by benzoate hydroxylation and deoxyribose oxidation; LADH counteracted the pro-oxidant effect of CAs by scavenging hydroxyl radicals. Captopril, dihydrolipo amide, dihydrolipoic acid, DL-dithiothreitol, GSSG, try-panothione and histidine effectively preserved LADH from oxidative damage, whereas N-acetylcysteine, N-(2-mercaptopropionylglycine) and lipoamide were less effective protectors. Catalase (though neither bovine serum albumin nor superoxide dismutase) protected LADH against the Cu(II)/H₂O₂/CAs systems. Dena tured catalase protected less than the native enzyme, its action possibly depending on Cu-binding. LADH in creased and Captopril inhibited epinephrine oxidation by Cu(II)/H₂O₂ and Cu(II). The summarized evidence supports the following steps for LADH inactivation: (1) reduction of LADH linked-Cu(II) to Cu(I) by CAs; (2) production of HO^* from H₂O₂ by LADH-linked Cu(I) (Haber-Weiss reaction) and (3) oxidation of aminoacid residues at the: enzyme active site by site-specifically generated HO^* radicals. Hydrogen peroxide formation from CAs autoxidation may contribute to LADH inactivation.     

Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.     

Iron(II) and hydrogen peroxide detoxification by human H-chain ferritin. An EPR spin-trapping study

Overexpression of human H-chain ferritin (HuHF) is known to impart a degree of protection to cells against oxidative stress and the associated damage to DNA and other cellular components. However, whether this protective activity resides in the protein's ability to inhibit Fenton chemistry as found for Dps proteins has never been established. Such inhibition does not occur with the related mitochondrial ferritin which displays much of the same iron chemistry as HuHF, including an Fe(II)/H(2)O(2) oxidation stoichiometry of approximately 2:1. In the present study, the ability of HuHF to attenuate hydroxyl radical production by the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + OH(-) + *OH) was examined by electron paramagnetic resonance (EPR) spin-trapping methods. The data demonstrate that the presence of wild-type HuHF during Fe(2+) oxidation by H(2)O(2) greatly decreases the amount of .OH radical produced from Fenton chemistry whereas the ferroxidase site mutant 222 (H62K + H65G) and human L-chain ferritin (HuLF) lack this activity. HuHF catalyzes the pairwise oxidation of Fe(2+) by the detoxification reaction [2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH(core) + 4H(+)] that occurs at the ferroxidase site of the protein, thereby preventing the production of hydroxyl radical. The small amount of *OH radical that is produced in the presence of ferritin (相似文献   

Inactivation of Lipoamide Dehydrogenase by Cobalt(II) and Iron(II) Fenton Systems: Effect of Metal Chelators, Thiol Compounds and Adenine Nucleotides

Fe(II)-and Co(II)-Fenton systems (FS) inactivated the lipoamide reductase activity but not the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). The Co(II) system was the more effective as LADH inhibitor. Phosphate ions enhanced the Fe(II)-FS activity. EDTA, DETAPAC, DL-histidine, DL-cysteine, glutathione, DL-dithiothreitol, DL-lipoamide, DL-thioctic acid, bathophenthroline, trypanothione and ATP, but not ADP or AMP, prevented LADH inactivation. Reduced disulfide compounds were more effective protectors than the parent compounds. Mg ions counteracted ATP protective action. Glutathione and DL-dithiothreitol partially restored the lipoamide dehydrogenase activity of the Fe(II)-FS-inhibited LADH. DL-histidine exerted a similar action on the Co(II)-FS-inhibited enzyme. Ethanol, mannitol and benzoate did not prevent LADH inactivation by the assayed Fenton systems and, accordingly, it is postulated that site-specific generated HO'radicals were responsible for LADH inactivation. With the Co(II)-FS, oxygen reactive species other than HO, might contribute to LADH inactivation.     

Hydrogen peroxide-mediated Cu,Zn-superoxide dismutase fragmentation: protection by carnosine, homocarnosine and anserine

The fragmentation of human Cu,Zn-superoxide dismutase (SOD) was observed during incubation with H(2)O(2). Hydroxyl radical scavengers such as sodium azide, formate and mannitol protected the fragmentation of Cu,Zn-SOD. These results suggested that *OH was implicated in the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation. Carnosine, homocarnosine and anserine have been proposed to act as anti-oxidants in vivo. We investigated whether three compounds could protect the fragmentation of Cu,Zn-SOD induced by H(2)O(2). The results showed that carnosine, homocarnosine and anserine significantly protected the fragmentation of Cu,Zn-SOD. All three compounds also protected the loss of enzyme activity induced by H(2)O(2). Carnosine, homocarnosine and anserine effectively inhibited the formation of *OH by the Cu,Zn-SOD/H(2)O(2) system. These results suggest that carnosine and related compounds can protect the hydrogen peroxide-mediated Cu,Zn-SOD fragmentation through the scavenging of *OH.     

Generation of *OH initiated by interaction of Fe2+ and Cu+ with dioxygen; comparison with the Fenton chemistry

Iron and copper toxicity has been presumed to involve the formation of hydroxyl radical (*OH) from H2O2 in the Fenton reaction. The aim of this study was to verify that Fe2+-O2 and Cu+-O2 chemistry is capable of generating *OH in the quasi physiological environment of Krebs-Henseleit buffer (KH), and to compare the ability of the Fe2+-O2 system and of the Fenton system (Fe2+ + H2O2) to produce *OH. The addition of Fe2+ and Cu+ (0-20 microM) to KH resulted in a concentration-dependent increase in *OH formation, as measured by the salicylate method. While Fe3+ and Cu2+ (0-20 microM) did not result in *OH formation, these ions mediated significant *OH production in the presence of a number of reducing agents. The *OH yield from the reaction mediated by Fe2+ was increased by exogenous Fe3+ and Cu2+ and was prevented by the deoxygenation of the buffer and reduced by superoxide dismutase, catalase, and desferrioxamine. Addition of 1 microM, 5 microM or 10 microM Fe2+ to a range of H2O2 concentrations (the Fenton system) resulted in a H2O2-concentration-dependent rise in *OH formation. For each Fe2+ concentration tested, the *OH yield doubled when the ratio [H2O2]:[Fe2+] was raised from zero to one. In conclusion: (i) Fe2+-O2 and Cu+-O2 chemistry is capable of promoting *OH generation in the environment of oxygenated KH, in the absence of pre-existing superoxide and/or H2O2, and possibly through a mechanism initiated by the metal autoxidation; (ii) The process is enhanced by contaminating Fe3+ and Cu2+; (iii) In the presence of reducing agents also Fe3+ and Cu2+ promote the *OH formation; (iv) Depending on the actual [H2O2]:[Fe2+] ratio, the efficiency of the Fe2+-O2 chemistry to generate *OH is greater than or, at best, equal to that of the Fe2+-driven Fenton reaction.     

Oxidative inactivation of an extramitochondrial acetyl-CoA hydrolase by autoxidation of L-ascorbic acid

The activity of acetyl-CoA hydrolase (dimeric form) purified from the supernatant fraction of rat liver was shown to have a half-life (t1/2) of 3 min at 0 degree C, but to stable at 37 degrees C (t1/2 = 34 h) [Isohashi, F., Nakanishi, Y. & Sakamoto, Y. (1983) Biochemistry 22, 584-590]. Incubation of the purified enzyme with L-ascorbic acid (AsA) at 37 degrees C resulted in inactivation of the enzyme (t1/2 = 90 min at 2 mM AsA). The extent of inactivation was greatly enhanced by addition of transition metal ions (Cu2+, Fe2+, and Fe3+). Thiol reducing agents, such as reduced glutathione and DL-dithiothreitol, protected the hydrolase from inactivation by AsA. However, these materials did not restore the catalytic activity of the enzyme inactivated by AsA. When AsA solution containing Cu2+ was preincubated under aerobic conditions at 37 degrees C for various times in the absence of enzyme, and then aliquots were incubated with the enzyme solution for 20 min, remaining activity was found to decrease with increase in the preincubation time, reaching a minimum at 60 min. However, further preincubation reduced the potential for inactivation. Catalase, a hydrogen peroxide (H2O2) scavenger, almost completely prevented inactivation of the enzyme by AsA plus Cu2+. Superoxide dismutase and tiron, which are both superoxide (O2-) scavengers, also prevented inactivation of the enzyme. A high concentration of mannitol, a hydroxyl radical (OH) scavenger, partially protected the enzyme from inactivation. These results suggest that inactivation of the enzyme by AsA in the presence of Cu2+ was due to the effect of active oxygen species (H2O2, O2-, OH) that are known to be autoxidation products of AsA. Valeryl-CoA, a competitive inhibitor of acetyl-CoA hydrolase, greatly protected the enzyme from inactivation by AsA plus Cu2+, but ATP and ADP, which are both effectors of this enzyme, had only slight protective effects. These results suggest that inactivation of this enzyme by addition of AsA plus Cu2+ was mainly due to attack on its active site.     

Metabolic mechanisms of methanol/formaldehyde in isolated rat hepatocytes: carbonyl-metabolizing enzymes versus oxidative stress

Methanol (CH(3)OH), a common industrial solvent, is metabolized to toxic compounds by several enzymatic as well as free radical pathways. Identifying which process best enhances or prevents CH(3)OH-induced cytotoxicity could provide insight into the molecular basis for acute CH(3)OH-induced hepatoxicity. Metabolic pathways studied include those found in 1) an isolated hepatocyte system and 2) cell-free systems. Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques demonstrated that CH(3)OH had little toxicity towards rat hepatocytes in 95% O(2), even at 2M concentration, whereas 50 mM was the estimated LC(50) (2h) in 1% O(2), estimated to be the physiological concentration in the centrilobular region of the liver and also the target region for ethanol toxicity. Cytotoxicity was attributed to increased NADH levels caused by CH(3)OH metabolism, catalyzed by ADH1, resulting in reductive stress, which reduced and released ferrous iron from Ferritin causing oxygen activation. A similar cytotoxic mechanism at 1% O(2) was previous found for ethanol. With 95% O(2), the addition of Fe(II)/H(2)O(2), at non-toxic concentrations were the most effective agents for increasing hepatocyte toxicity induced by 1M CH(3)OH, with a 3-fold increase in cytotoxicity and ROS formation. Iron chelators, desferoxamine, and NADH oxidizers and ATP generators, e.g. fructose, also protected hepatocytes and decreased ROS formation and cytotoxicity. Hepatocyte protein carbonylation induced by formaldehyde (HCHO) formation was also increased about 4-fold, when CH(3)OH was oxidized by the Fenton-like system, Fe(II)/H(2)O(2), and correlated with increased cytotoxicity. In a cell-free bovine serum albumin system, Fe(II)/H(2)O(2) also increased CH(3)OH oxidation as well as HCHO protein carbonylation. Nontoxic ferrous iron and a H(2)O(2) generating system increased HCHO-induced cytotoxicity and hepatocyte protein carbonylation. In addition, HCHO cytotoxicity was markedly increased by ADH1 and ALDH2 inhibitors or GSH-depleted hepatocytes. Increased HCHO concentration levels correlated with increased HCHO-induced protein carbonylation in hepatocytes. These results suggest that CH(3)OH at 1% O(2) involves activation of the Fenton system to form HCHO. However, at higher O(2) levels, radicals generated through Fe(II)/H(2)O(2) can oxidize CH(3)OH/HCHO to form pro-oxidant radicals and lead to increased oxidative stress through protein carbonylation and ROS formation which ultimately causes cell death.     

Egg yolk phosvitin inhibits hydroxyl radical formation from the fenton reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical ((.-)OH) formation from H(2)O(2) in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited (.-)OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the (.-)OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H(2)O(2). These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles

Na-Ca exchange activity in bovine cardiac sarcolemmal vesicles was stimulated up to 10-fold by preincubating the vesicles with 1 microM FeSO4 plus 1 mM dithiothreitol (DTT) in a NaCl medium. The increase in activity was not reversed upon removing the Fe and DTT. Stimulation of exchange activity under these conditions was completely blocked by 0.1 mM EDTA or o-phenanthroline; this suggests that the production of reduced oxygen species (H2O2, O2-.,.OH) during Fecatalyzed DTT oxidation might be involved in stimulating exchange activity. In agreement with this hypothesis, the increase in exchange activity in the presence of Fe-DTT was inhibited 80% by anaerobiosis and 60% by catalase. H2O2 (0.1 mM) potentiated the stimulation of Na-Ca exchange by Fe-DTT under both aerobic and anaerobic conditions; H2O2 also produced an increase in activity in the presence of either FeSO4 (1 microM) or DTT (1 mM), but it had no effect on activity by itself. Superoxide dismutase did not block the effects of Fe-DTT on exchange activity; however, the generation of O2-. by xanthine oxidase in the presence of an oxidizable substrate stimulated activity more than 2-fold. Hydroxyl radical scavenging agents (mannitol, sodium formate, sodium benzoate) did not attenuate the stimulation of activity observed with Fe-H2O2. Exchange activity was also stimulated by the simultaneous presence of glutathione (GSH; 1-2 mM) and glutathione disulfide (GSSG; 1-2 mM). Neither GSH nor GSSG was effective by itself and either 0.1 mM EDTA or o-phenanthroline blocked the effects on transport activity of the combination of GSH + GSSG. Treatment of the GSH and GSSG solutions with Chelex ion-exchange resin to remove contaminating transition metal ions reduced (by 40%) the degree of stimulation observed with GSH + GSSG. Full stimulating activity was restored to the Chelex-treated GSH and GSSG solutions by the addition of 1 microM Fe2+; Cu2+ was less effective than Fe2+ whereas Co2+ and Mn2+ were without effect. In the presence of 1 microM Fe2+, GSH alone produced a slight increase in transport activity, but this was markedly enhanced by the addition of Chelex-treated GSSG. The results indicate that stimulation of exchange activity requires the presence of both a reducing agent (DTT, GSH, O-.2, or Fe2+) and an oxidizing agent (H2O2, GSSG, and perhaps O2) and that the effects of these agents are mediated by metal ions (e.g. Fe2+).(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of Fenton's reagent by cellobiose oxidase from cellulolytic cultures of Phanerochaete chrysosporium.

The reduction of dioxygen by cellobiose oxidase leads to accumulation of H2O2, with either cellobiose or microcrystalline cellulose as electron donor. Cellobiose oxidase will also reduce many Fe(III) complexes, including Fe(III) acetate. Many Fe(II) complexes react with H2O2 to produce hydroxyl radicals or a similarly reactive species in the Fenton reaction as shown: H2O2 + Fe2+----HO. + HO- + Fe3+. The hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid is a standard test for hydroxyl radicals. Hydroxylation was observed in acetate buffer (pH 4.0), both with Fe(II) plus H2O2 and with cellobiose oxidase plus cellobiose, O2 and Fe(III). The hydroxylation was suppressed by addition of catalase or the absence of iron [Fe(II) or Fe(III) as appropriate]. Another test for hydroxyl radicals is the conversion of deoxyribose to malondialdehyde; this gave positive results under similar conditions. Further experiments used an O2 electrode. Addition of H2O2 to Fe(II) acetate (pH 4.0) or Fe(II) phosphate (pH 2.8) in the absence of enzyme led to a pulse of O2 uptake, as expected from production of hydroxyl radicals as shown: RH+HO.----R. + H2O; R. + O2----RO2.----products. With phosphate (pH 2.8) or 10 mM acetate (pH 4.0), the O2 uptake pulse was increased by Avicel, suggesting that the Avicel was being damaged. Oxygen uptake was monitored for mixtures of Avicel (5 g.1-1), cellobiose oxidase, O2 and Fe(III) (30 microM). An addition of catalase after 20-30 min indicated very little accumulation of H2O2, but caused a 70% inhibition of the O2 uptake rate. This was observed with either phosphate (pH 2.8) or 10 mM acetate (pH 4.0) as buffer, and is further evidence that oxidative damage had been taking place, until the Fenton reaction was suppressed by catalase. A separate binding study established that with 10 mM acetate as buffer, almost all (98%) of the Fe(III) would have been bound to the Avicel. In the presence of Fe(III), cellobiose oxidase could provide a biological method for disrupting the crystalline structure of cellulose.